61 results about "Nicking enzyme" patented technology

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

An artificial nicking enzyme comprising two domains ND1 and ND1n linked via an ND1 linker, wherein ND1 is at least one polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 98, as well as variants thereof which are polypeptides comprising an amino acid sequence wherein the amino acids corresponding to the 66th and 83rd amino acids of the amino acid sequence set forth in SEQ ID NO: 98 are aspartic acid, and, ND1n is at least one polypeptide selected from the group consisting of (an) a polypeptide comprising an amino acid sequence which is a substituted amino acid wherein the 66th aspartic acid and / or the 83rd aspartic acid of the amino acid sequence set forth in SEQ ID NO: 98 is substituted with another arbitrary amino acid, as well as variants thereof which are polypeptides comprising an amino acid sequence wherein the amino acid corresponding to the 66th aspartic acid and / or the 83rd aspartic acid of the amino acid sequence set forth in SEQ ID NO: 98 is a substituted amino acid substituted with an arbitrary amino acid other than aspartic acid, the nicking enzyme.

The invention relates to a method for simultaneously amplifying a plurality of target nucleic acid sequences in a sample, said method having the step of: (S300) amplifying the target nucleic acid sequences by means of an isothermal strand displacement amplification process using pairs of flanked primers, a superprimer (10), and at least one first nicking enzyme. One pair of flanked primers is used for each target nucleic acid sequence to be amplified, and the pair of flanked primers is specific to the associated target nucleic acid sequence, each pair of flanked primers having a flanked forward primer and a flanked reverse primer; each flanked forward primer has a target-specific portion at the 3' end thereof and a a non-specific flank portion at the 5' end thereof, the target-specific portion being complementary to a portion of the associated target nucleic acid sequence; each flanked reverse primer has a target-specific portion at the 3' end thereof and a non-specific flank portion at the 5' end thereof, said target-specific portion being complementary to a portion of the associated target nucleic acid sequence; and the superprimer (10) has a binding portion (12) at the 3' end thereof and a recognition portion (14) at the 5' end thereof, said recognition portion (14) having a recognition sequence for the first nicking enzyme.

Provided herein are gene editing systems and compositions directed to effectuate in vivo edits in the LPA gene. Treatment or prevention of cardiovascular disease through disruption of the production of apo(a) through genetic editing and the reduction of the blood lipoprotein(a) [Lp(a)] concentration is disclosed herein. Disclosed are nickase-based gene editing systems designed to effectuate the installation of insertions and / or deletions (indel variants) and / or non-synonymous variants in the coding sequence of LPA. The nickase-based gene editing systems generally comprise one or more mRNAs that encode one or more nickases and a plurality of guide oligonucleotides (e.g., gRNAs) and may be delivered in vivo to a mammalian subject in need thereof via a suitable delivery system, such as lipid nanoparticles (LNPs) (with or without GalNAc targeting moieties) intravenously, or otherwise, administered to a patient as potentially a once-and-done therapeutic. The manufacturing, use, and formulation of the gene editing systems and compositions are also disclosed.

This invention pertains to fusion protein mutants comprising a Prime Editing enzyme having a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant), wherein the fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

Provided are systems for strand-specific editing of DNA, including mitochondrial DNA in humans. The systems provided herein comprise a nickase and a single-stranded (ss) DNA deaminase each, or together, associated with a double-stranded DNA binding polypeptide such that DNA editing occurs in an editing region. Also provided herein are components of the systems for editing DNA taught herein, and methods of use thereof.

A modified pegRNA Cas9 nickase system that maintains potent on-target editing activity but has reduced off-target editing activity relative to wild-type Cas, and methods of production and use.

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

The present application relates to the field of biotechnology, and particularly relates to a method for identifying a cleavage site of a nucleic acid nicking enzyme. The present application discloses a method for identifying a cleavage site of a nicking enzyme, which comprises the following steps: obtaining nucleic acid sequence data before and after enzyme cleavage by using mNGS sequencing, analyzing second-generation sequencing data by using Bowite2 and samtools software, and obtaining the cleavage site of the nicking enzyme according to single-base sequencing depth and analysis. The present application does not need to determine the sequence of the nucleic acid to be cleaved, has short time consumption and low budget, and has a good application prospect.

This application belongs to the field of biotechnology and relates to an efficient and specific editing system, method, and application for A-G base substitution. The fusion protein described in this application comprises, from the N-terminus to the C-terminus, a first SaCas9 nickase fragment, a chimeric deaminase fragment, and a second SaCas9 nickase fragment. The deaminase is selected from adenosine deaminase or a variant thereof, specifically ecTadA8e. The amino acid sequence of ecTadA8e is shown in SEQ ID NO. 20, or has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 20, and possesses the function or activity of ecTadA8e. The fusion protein provided in this application, combined with the corresponding guide RNA, can efficiently and specifically replace the base A with G at the target site, providing an effective tool for repairing pathogenic mutations, studying gene function, and improving cell function, and has promising application prospects.

This invention discloses a method and kit for detecting RNA using a three-stage amplification melting curve based on isothermal self-circulation. The method includes designing a first primer, a second primer, and a circular amplification probe targeting the target nucleic acid sequence. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end. The circular amplification probe is a circular DNA segment whose sequence consists of three parts: the first part completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence from the second primer; the second part is a self-cleaving DNase sequence. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed, achieving exponential signal amplification of the target nucleic acid through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification.

A chemically modified polynucleotide encoding a fusion protein, wherein the chemically modified polynucleotide comprises a sequence of SEQ ID NO: 701 is claimed wherein the fusion comprises a Cas9 nickase and and deaminase, such as an adenosine deamnase TadA. and which further comprises a protospacer found on a gene encoding Angiopoietin-like 3 protein (ANGPTL3). The chemical modifications may include phosphorothioate linkages and 2'-O-methyl modfied nucleosides. Use of such fusions in treating cardiovascular conditions is further claimed. [Figure 3]

The invention provides sgRNA (small guide ribonucleic acid) and target DNA (deoxyribonucleic acid) for detecting the activity of SpCas9 D10A Nickase and a detection method. A reaction system capable of expressing the function and a quantitative detection method (IEX-HPLC method) are established by simulating the enzyme activity function of a base editing enzyme incision, so that the requirements on experimental animals and cells are remarkably reduced, the detection period is shortened, and the 3R (reduction, optimization and substitution) principle of modern scientific research is met; meanwhile, the invention also provides an efficient and practical analysis method for the quality characteristic analysis work of the base editing enzyme, the convenience of quality detection in gene editing product production and research is improved, and the method has a large-scale commercial application prospect.

The invention relates to the technical field of agriculture, in particular to an efficient ABE base editing tool based on Cas12i3 and application of the efficient ABE base editing tool. The Cas12i3-based efficient ABE base editing tool provided by the invention comprises a protein expression cassette and an RNA (Ribonucleic Acid) expression cassette, the protein expression cassette comprises a promoter, a 2 * BP NLS nuclear localization signal fragment, a 2 * adenine deaminase TadA8e, a connecting peptide, a Cas12i3 notch enzyme fragment and a terminator, the RNA expression cassette comprises a promoter, a crRNA sequence and a terminator; wherein the crRNA sequence is composed of a DR sequence and a target specific spacer sequence. The tool realizes efficient and specific single base substitution of A-to-G in a rice genome by utilizing the targeted binding characteristic of Cas12i3 and the efficient deamination activity of 2 * TadA8e, has the advantages of high editing efficiency, low off-target rate, wide application range and the like, provides an efficient and accurate technical means for plant functional genome research and molecular breeding, and has a wide application prospect. The creation of excellent rice varieties can be accelerated.

The present invention relates to the field of genomic engineering. In particular, a chimeric prime editing (cPE) system is disclosed comprising elements including, but not limited to a Cas9 nickase (nCas9) / high fidelity nucleotide polymerase (HFNTPol) RNA, one or more single guide RNAs (sgRNAs), and a chimeric prime editor template oligonucleotide (cpetODN) comprising a deoxyribonucleic acid nucleotide polymerase template (NPT) and a primer binding site. For example, the sgRNA and the cpetODN are ligated into a single oligonucleotide. Alternatively, the sgRNA and the cpetODN are free and independent molecules (e.g., modular). This cPE system results in precise and efficient genome editing in cells and in adult mouse liver which is advantageous over conventional sgRNA prime editor fusion constructs. This flexible and modular system is an improvement in the art to obtain precise genome editing.

The application belongs to the technical field of biological medicine, and discloses a gene leading editing system, which comprises a carrier, the carrier contains a gene coding pegRNA and a gene coding fusion protein, the pegRNA comprises sgRNA, reverse transcription template RTT, primary binding site PBS and RNA stem loop structure, and the fusion protein is a fusion protein of Cas9 nickase or a variant thereof and reverse transcriptase. The application develops sPEs by adding an RNA neck ring structure to the 3' end of the pegRNA, develops tPEs by allowing the 3' end of the pegRNA to bind to Cas9, so as to improve the stability of the Cas9 / pegRNA complex, and further improve the editing efficiency of different genomic sites in different cells.

In one aspect, the disclosure relates to a system for site-specific modification of a double­stranded target DNA sequence, the system including at least: a fusion protein comprising an RNA-binding nickase protein and a DNA-dependent DNA polymerase, and a chimeric guide nucleic acid sequence comprising a guide RNA sequence that interacts with the RNA-binding nickase protein and a single-stranded DNA template, wherein the single-stranded DNA template comprises a modified sequence for insertion into a second strand of the double-stranded target DNA sequence and a primer binding site. Also disclosed is a method for site-specific modification of a double-stranded target DNA sequence in a cell. In another aspect, the present disclosure is directed to a fusion protein including an RNA-binding nickase protein and a DNA-dependent DNA polymerase, wherein one or both of the nicakse protein and the DNA polymerase can include one or more mutations to optimize function and prevent off-target effects.

The invention discloses a precise editing method of a gamma-PGA high-yield strain based on CRISPR-Cas9n, and relates to the technical field of high-yield strains. According to the invention, a CRISPR-Cas9n double-nickase system is adopted, and adjacent DNA nicks are generated through the synergistic effect of double sgRNA, so that the non-specific cutting risk is greatly reduced; the double sgRNA targeting design is combined with a homologous recombination template, so that directional editing of key genes is realized, the homologous recombination efficiency reaches 75 + / -5%, and target modification is ensured to be stably integrated into a genome; by virtue of a PEG-mediated protoplast conversion method, the limitation of low efficiency of traditional electrotransfection is overcome, and the CRISPR carrier is rapidly removed in combination with a temperature-sensitive plasmid elimination system, so that cytotoxicity caused by continuous expression is avoided; under optimized fermentation conditions, the gamma-PGA yield of the edited strain reaches 15 + / -2g / L and is increased by 200% compared with that of the original strain.

Gene amplifications are an oncogenic driver utilized by many forms of cancer in tumor development or treatment relapse. Promoting genomic instability or the proclivity to propagate genomic alterations through acquired defects in DNA repair machinery, replication licensing, or cell cycle control, gene amplifications not only drive oncogenesis, but also afford an opportunity for therapeutic exploitation. Here, CRISPR-Cas9 nickases are disclosed which selectively promote cancer cell death in a gene amplification-dependent manner. For example, CRISPR- Cas9 nickases generate a lethal number of highly toxic single-ended double-strand breaks within the genome of proliferating cancer cells harboring amplified genomic loci during DNA replication. Cas9 nickases may serve as a tumor-selective therapeutic that mitigates the collateral damage observed with conventional chemoradiotherapies and avoids chemoresistance.

We described herein are click edits, which are a series of genome editing methods that combine DNA nicking enzymes, including but not limited to RNA programmable CRISPR nicking enzymes, with DNA ligases or DNA polymerases to perform many different genome edits. Edits of interest are encoded on nucleic acid templates or substrates (optionally'click nucleic acids' (clkNA) or'click DNA '(clkDNA) as described herein) that can be provided in trans form or recruited preferentially to a target site via a variety of nucleic acid telomere methods (optionally HUH family endonucleases (HUHe), telomere binding proteins (TBP), or DNA binding proteins or RNA binding proteins).

The invention provides a desoxyribonucleic acid synthesis method, a desoxyribonucleic acid synthesis template and a kit, the method comprises: combining a primer and a desoxyribonucleic acid synthesis template to obtain a first sub-desoxyribonucleic acid, the primer being a first nucleic acid sequence, the combination region of the desoxyribonucleic acid synthesis template comprising a second nucleic acid sequence, the first sub-desoxyribonucleic acid being a first sub-desoxyribonucleic acid; the first nucleic acid sequence and the second nucleic acid sequence are subjected to base pairing; desoxyribonucleic acid polymerase is added; the 3'terminal of the first nucleic acid sequence forms an extension sequence in an extension region of the deoxyribonucleic acid synthesis template to obtain a second sub-deoxyribonucleic acid, the extension region comprises a third nucleic acid sequence and a fourth nucleic acid sequence, the third nucleic acid sequence and the fourth nucleic acid sequence are paired in base, and the extension sequence and the third nucleic acid sequence have the same structure; according to the synthesis method, the synthesis speed can be increased, the sequence length of the deoxyribonucleic acid can be increased, and the synthesis cost can be reduced.

The present invention relates to a modified pegRNA (prime editing guide RNA) advantageous for 5' flap cleavage and a method for enhancing prime editing efficiency using said modified pegRNA. More specifically, the invention relates to a modified pegRNA having seven or more nucleotides added to the 5' upstream of an RTT (reverse transcription template) sequence that bind to an endogenous 5' overhang single-stranded DNA generated by Cas9 nickase, and a gene editing composition containing said modified pegRNA. The modified pegRNA of the present invention has the effect of enhancing prime editing efficiency by increasing the cleavage of an endogenous 5' DNA flap that does not contain the desired editing sequence.

The invention relates to the field of gene engineering, and discloses a pilot editing system, a recombinant strain containing the pilot editing system, application of the recombinant strain and a gene editing method. The system comprises a coding gene of nicking enzyme nCas9, a coding gene of reverse transcriptase RT and pegRNA. Wherein the coding gene of the nicking enzyme nCas9 and the coding gene of the reverse transcriptase RT are connected through a Linker sequence, and the nucleotide sequence of the Linker sequence is as shown in SEQ ID NO. 1; the pegRNA comprises a gibberella zeylanica endogenous promoter 5SrRNA, sgRNA, a spacer sequence spacer, a primer binding site PBS (Phosphate Buffer Solution) and a reverse transcription template RT template; the reverse transcription template RT template carries an insertion sequence, and the insertion sequence is used for destroying the expression of a target gene. The method realizes efficient, accurate and safe gene editing of gibberella zeylanica, and has important scientific research and application values.

Provided herein are gene editing systems and compositions relating to enabling in vivo editing in the LPA gene. Disclosed herein are treatment or prevention of cardiovascular diseases by disruption of apo (a) production and reduction of blood lipoprotein (a) [Lp (a)] concentration via genetic editing. A nickase-based gene editing system is disclosed that aims to enable insertion and / or deletion (insertion deletion variants) and / or installation of non-synonymous variants in the coding sequence of the LPA. Nicking enzyme-based gene editing systems generally comprise one or more mRNAs encoding one or more nicking enzymes and a plurality of guide oligonucleotides (e.g., gRNAs), and may be delivered in vivo to a mammalian subject in need thereof via a suitable delivery system, such as lipid nanoparticles (LNPs) with or without a GalNAc targeting moiety, such as a GalNAc targeting moiety. The delivery system is administered intravenously or otherwise as a potential disposable therapy to a patient. Preparation, use and formulation of the gene editing system and composition are also disclosed.

Provided herein are gene editing systems and compositions directed to effectuate in vivo edits in the AGT gene. Treatment or prevention of a disease, such as hypertension, through disruption of the production of angiotensinogen via genetic editing is disclosed herein. Disclosed are nickase-based gene editing systems designed to effectuate the installation of insertions and / or deletions (indel variants) and / or non-synonymous variants in the coding sequence of AGT. The nickase-based gene editing systems generally comprise one or more mRNAs that encode one or more nickases and a plurality of guide oligonucleotides (e.g., gRNAs) and may be delivered in vivo to a mammalian subject in need thereof via a suitable delivery system, such as lipid nanoparticles (LNPs) (with or without GalNAc targeting moieties) intravenously, or otherwise, administered to a patient as potentially a single-course or once-and-done therapeutic. The manufacturing, use, and formulation of the gene editing systems and compositions are also disclosed.

The disclosure relates generally to gene editing systems comprising reverse transcriptases and fusion proteins of reverse transcriptases with nickases or nucleases, methods of making such reverse transcriptases and fusion proteins, and methods of using such reverse transcriptases and fusion proteins for site directed genome editing in cells.

The present invention relates to a base editing method for plant DNA, the method comprising a step of expressing a base editing composition in a target plant, plant cell or protoplast, the base editing composition comprising one or more DNA binding proteins and one or more zymoproteins, or comprising a polynucleotide encoding said proteins. The invention is used for editing bases in nuclear DNA or organelle DNA of plants, especially for editing bases in organelle DNA of plants such as chloroplast or mitochondria. The base editing composition can be used for realizing targeted editing from adenine (A) base to guanine (G), targeted editing from cytosine (C) base to thymine (T) base or a method for realizing the two editing methods at the same time in plant DNA (Deoxyribose Nucleic Acid). In addition, the present invention relates to a plant cell, plant or seed implementing such base editing.

The technology described herein relates to new nuclease and nickase fusions of new CRISPR / Cas systems and their use as a biocontainment kill-switch in modified and / or engineered bacteria.